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Erythropoietin-derived short peptide and its mimics as immuno/inflammatory modulators

Erythropoietin-derived short peptide and its mimics as immuno/inflammatory modulators description/claims

This application claims priority to U.S. provisional application No. 60/676,592, filed Apr. 29, 2005, the entire contents of which are incorporated by reference herein.

The present invention relates to erythropoietin-derived short peptides and their mimics that protect against tissue damage in animals having diverse forms of neural and non-neural organ system injury.

Erythropoietin and its Non-Hematopoietic Activities

Erythropoietin (EPO), a 165 amino acid glycoprotein hormone, was identified initially as a hematopoietic growth factor and has been used extensively for the treatment of anemia in humans. Whole molecule EPO received considerable attention recently because it may have broad neuroprotective capabilities following CNS injury [Brines, M. L., et al., Proc Natl Acad Sci USA, 97: 10526-31 (2000); Siren, A. L. and Ehrenreich, H., Eur Arch Psychiatry Clin Neurosci, 251: 179-84 (2001); Buemi, M., et al., J. Neuropathol Exp Neurol, 62: 228-36 (2003); Li, W., et al., Ann Neurol, 56: 767-77 (2004); Sakanaka, M., et al., Proc Natl Acad Sci USA, 95: 4635-40 (1998)]. Therapeutic effects of exogenously administered EPO on several diverse forms of neurologic injury, including occlusive cerebral vascular disease, acute brain trauma, epilepsy, and an autoimmune model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE), have been tested and the degree of neurologic impairment was significantly reduced [(Brines, M. L. et al., Proc Natl Acad Sci USA, 97: 10526-31 (2000); Li, W. et al., Ann Neurol, 56: 767-77 (2004); Tsai, P. T., et al., J Neurosci, 26: 1269-74 (2006); Buemi, M., et al., Clin Sci (Lond), 103: 275-82 (2002)]. However, long-term EPO therapy remains significantly limited in non-anemic patients with neurological injury because EPO treatment may overly stimulate erythropoiesis. To overcome this concern, EPO therapy would have to be limited to very short term use. Other EPO molecular preparations, such as an asialo-form of EPO, carbamylated EPO (CEPO), or certain EPO mutants, have been shown to be neuroprotective in animals following experimental traumatic spinal cord injury or acute stroke without provoking an increase in red cell mass [Erbayraktar, S., et al., Proc Natl Acad Sci USA, 100: 6741-46 (2003); Leist, M., et al., Science, 305: 239-42 (2004); Mun, K. C. and Golper, T. A. Blood Purif, 18: 13-17 (2000); Brines, M., et al., Proc Natl Acad Sci USA, 101: 14907-12 (2004). A short 17 amino acid EPO-derived linear peptide also was reported to have neuroprotective effects in cell culture, but its in vivo biologic effects were not certain [Campana, W. M., et al., Int'l J Mol Med, 1: 235-41 (1998). Taken all together, the evidence suggests that specific functional and structural domains may co-exist within the full 165 amino acid EPO molecule.

The hematopoietic effect of EPO is mediated by binding and inducing dimerization of two molecules of the EPO receptor (EpoR) on the cell surface [Watowich, S. S., et al., Mol Cell Biol, 14: 3535-49 (1994)]. The EpoR belongs to a cytokine receptor superfamily that is also related to the cytokines granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukins 2-7 and ciliary neurotrophic factor (CNTF). The signaling pathway involves the autophosphorylation and activation of the Janus family protein tyrosine kinase, JAK-2, which further activates additional signaling proteins including STAT5, Ras-mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K). Studies on structure activity relationships of EPO have identified regions and amino acids essential for binding to the erythropoietin receptor (EpoR) [Livnah, O., et al., Science, 273: 464-71 (1996); Wrighton, N. C., et al., Science, 273: 458-64 (1996); Wen, D., J Biol Chem, 269: 22839-46 (1994)]. Studies in which recombinant EPO and EPO mutants have been tested for their biological effects in a variety of animal models have suggested that the neuroprotection mediated by EPO might not occur through a conventional interaction between EPO and classic EpoR. The common β receptor (βcR) or CD131, which is also an important component for other ligands including IL-3, IL-5 and GM-CSF, has been proposed to be a key subunit associated with the EpoR that is responsible for EPO mediated non-hematopoietic effects. Additional unknown receptor(s) also may play critical roles in the non-hematopoietic effects induced by chemically modified or mutant EPO. Based on the belief that there are at least two distinct functional peptide domains co-existing within the whole EPO molecule and that sequences and/or structures within the EPO-derived peptides will dictate their biological functions, small EPO-derived peptides with different biological functions having neuroprotective and immunomodulatory activity have been identified and characterized herein.

EAE Animal Model and Multiple Sclerosis

Multiple sclerosis (MS) a disorder of unknown cause, is defined clinically by characteristic symptoms, signs and progression, and is defined pathologically by scattered areas of inflammation and demyelination affecting the brain, optic nerves and spinal cord white matter. It is widely believed that the pathogenesis of MS involves an immune-mediated inflammatory demyelinating process.

Experimental autoimmune encephalomyelitis (EAE) is a central nervous system inflammatory demyelinating disease involving acute injury to the brain and spinal cord white matter. This animal model has been used widely by many investigators to study disease pathogenesis and to explore new therapies for its human counterpart, multiple sclerosis (MS). Pathogenesis of both MS and EAE is believed to involve (1) activation of myelin reactive T cells; (2) upregulated expression of chemokines and adhesion molecules; (3) focal T cells and macrophage infiltration into the CNS white matter; and (4) demyelination and axonal injury and loss of neurological function [Trapp., B. et al., J Neuroimmunol, 98: 49-56 (1999)]. In both EAE and MS, activated T-lymphocytes specific for self-antigens present in myelin are linked to CNS inflammation and to the breakdown of the blood brain barrier to peripheral blood leukocytes and plasma proteins; this is predominantly restricted to myelin rich white matter area of the CNS [Bettelli, E., et al., J Exp Med, 197: 1073-81 (2003); Crawford, M. P., et al., Blood 103(11): 4222-31 (2004); Abdul-Majid, K. B., et al., J Neuroimmunol, 141: 10-19 (2003); Battistini, L., et al., Blood, 101: 4775-82 (2003).

EAE can be induced experimentally in genetically susceptible animals, such as mice, by immunization with immunodominant peptides from myelin proteins, such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocytes glycoprotein (MOG), emulsified in complete Freund's adjuvant followed by injection of pertussis toxin as an additional adjuvant for certain mouse strains [Li, W., et al., Ann Neurol, 56: 767-77 (2004). Disease development is variable from strain to strain. For example, in SJL/J mice, PLP or MBP induces a relapsing-remitting progression, whereas C57BL/6 mice immunized with MOG often develop a chronic form of disease.

A library of stabilized isolated EPO-derived peptides comprising about 7 to about 25 amino acids in length has been created and tested in vitro and in vivo for therapeutic efficacy. It has been shown that these short EPO-derived peptides are highly protective in mouse models of EAE, acute stroke, acute spinal cord and brain injury as well as arthritis by reversing and/or reducing manifestations of the associated disease. This protection is maintained during long term observation in EAE mice and was not associated with hematological side effects. These short peptides protect against tissue damage by modulating the immune-mediated inflammatory network, i.e. by reducing major histocompatibilty complex (MHC) class I and class II over-expression; by reducing inflammatory cytokines; and by suppressing antigen-specific T cell function in peripheral lymphoid tissue and brain tissue as well as in in vitro tissue culture assays. The peptide domain that is crucial for immunomodulating/anti-inflammation functions as well as a small peptide ring that plays an important role in maintaining the stability, without hampering biologic efficacy, of these small peptides have been identified. Moreover, addition of a small bicyclic compound, such as d-biotin, to the N- or C-terminal of linear cytokine-derived peptides, including, but not limited to, short EPO linear peptides, the stability of these peptides is increased without hampering their biologic activity. These newly developed small peptides hold immense potential for direct clinical application in treatment of central and peripheral nervous system diseases associated with injury including demyelinating diseases, traumatic injury and stroke. Since the beneficial effect of these peptides is not limited to neural tissue organs, they are broadly useful in the treatment of inflammatory/immune injury to non-neural organs of the body.

The present invention provides stabilized, isolated EPO-derived peptides and their mimics that protect against tissue damage in subjects having diverse forms of neural and non-neural organ system injury, pharmaceutical compositions containing the isolated EPO-derived peptides, methods for treating symptoms of a disease, disorder or condition having an inflammatory or an autoimmune component in a subject in need thereof, and methods for downregulating immune mediator activity in a subject in need thereof. In one embodiment of the present invention, a stabilized isolated EPO-derived peptide comprises an amino acid sequence comprising an N-terminal end and a C-terminal end and having a non-hematopoietic biological activity, wherein the non-hematopoietic biological activity of the stabilized EPO-derived peptide is stable when the peptide is stored at 4° C. In another embodiment, this EPO-derived peptide is stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or the C-terminal end of the peptide's amino acid sequence. In another embodiment, this stabilized isolated EPO-derived peptide is at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 27. In another embodiment, the stabilized, isolated EPO-derived peptide is stabilized by a disulfide bond formed between a sulfhydral group of a first amino acid residue and a sulfhydral group of a second amino acid residue along the peptide sequence. In another embodiment, the stabilized, isolated EPO-derived peptide comprises at least 6 amino acids having the peptide sequence XAEHYS, wherein X is a first amino acid residue containing a sulfhydral group and Y is a second amino acid residue containing a sulfhydral group, and wherein the sulfhydral group of the first amino acid residue is at an appropriate distance from the sulfhydral group of the second amino acid residue to form the disulfide bond with the sulfhydral group of the second amino acid residue within the peptide sequence, thus stabilizing the peptide. In another embodiment, the disulfide-bond-stabilized, isolated EPO-derived peptide is at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO; 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15. In another embodiment, the disulfide bond-stabilized isolated EPO-derived peptide is further stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or the C-terminal end of the peptide's amino acid sequence. In yet another embodiment, the EPO-derived peptide comprises at least 7 amino acids having the peptide sequence XAEHYS, wherein X is a first amino acid residue containing a sulfhydral group and Y is a second amino acid residue containing a sulfhydral group, and wherein the sulfhydral group of the first amino acid residue is at an appropriate distance from the sulfhydral group of the second amino acid residue to form the disulfide bond with the sulfhydral group of the second amino acid residue within the peptide sequence, thus stabilizing the peptide. In another embodiment, this disulfide bond-stabilized isolated EPO-derived peptide is at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 30. In another embodiment, this disulfide bond-stabilized EPO-derived peptide is further stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or the C-terminal end of the peptide's amino acid sequence. In another embodiment, the disulfide bond-stabilized EPO-derived peptide further stabilized by chemically adding a small bicyclic molecule is at least one peptide whose amino acid sequence is SEQ ID NO: 20. In yet another embodiment, the isolated stabilized EPO-derived peptide mimics the non-hematopoietic biological activity of at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO; 3, SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 27, AND SEQ ID NO: 30.

The present invention further provides a pharmaceutical composition for treating symptoms of a disease, disorder or condition having an inflammatory or autoimmune component in a subject in need thereof comprising a therapeutically effective amount of at least one stabilized isolated EPO-derived peptide, wherein the animals' red cell indices remain substantially at normal levels during treatment. In one embodiment, the disease, disorder or condition having an inflammatory or autoimmune component is at least one disease, disorder or condition selected from the group consisting of immune-mediated inflammation, an autoimmune disease, a demyelinating disease, an arthritis, an acute cerebrovascular injury, an acute spinal cord injury, an acute brain injury, and an acute cardiovascular injury. In another embodiment, the stabilized EPO-derived peptide of the composition comprises an amino acid sequence comprising an N-terminal end and a C-terminal end and has a non-hematopoietic biological activity, wherein the non-hematopoietic biological activity of the stabilized EPO-derived peptide is stable when the peptide is stored at 4° C. In another embodiment, the isolated stabilized EPO-derived peptide is stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or the C-terminal end of the peptide's amino acid sequence. In another embodiment, the isolated stabilized EPO-derived peptide of the composition is at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 27. In another embodiment, the stabilized EPO-derived peptide of the composition is stabilized by a disulfide bond formed between a sulfhydral group of a first amino acid residue and a sulfhydral group of a second amino acid residue along the peptide sequence. In another embodiment, the stabilized isolated EPO-derived peptide of the composition comprises at least 6 amino acids having the peptide sequence XAEHYS, wherein X is a first amino acid residue containing a sulfhydral group and Y is a second amino acid residue containing a sulfhydral group, and wherein the sulfhydral group of the first amino acid residue is at an appropriate distance from the sulfhydral group of the second amino acid residue to form the disulfide bond with the sulfhydral group of the second amino acid residue within the peptide sequence, thus stabilizing the peptide. In another embodiment, the isolated disulfide bond-stabilized EPO-derived peptide is at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO; 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15. In another embodiment, the isolated stabilized EPO-derived peptide of the composition comprises at least 7 amino acids having the peptide sequence XAEHYS, wherein X is a first amino acid residue containing a sulfhydral group and Y is a second amino acid residue containing a sulfhydral group, and wherein the sulfhydral group of the first amino acid residue is at an appropriate distance from the sulfhydral group of the second amino acid residue to form the disulfide bond with the sulfhydral group of the second amino acid residue within the peptide sequence, thus stabilizing the peptide. In another embodiment, the isolated disulfide bond-stabilized EPO-derived peptide of the composition is at least one peptide whose amino acid sequence is SEQ ID NO: 30. In another embodiment, the isolated disulfide-bond stabilized EPO-derived peptide of the composition is further stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or The C-terminal end of the peptide's amino acid sequence. In another embodiment, the isolated disulfide bond-stabilized EPO-derived peptide further stabilized by chemically adding the small bicyclic molecule lis at least one peptide whose amino acid sequence is SEQ ID NO: 20. In another embodiment, the isolated stabilized EPO-derived peptide of the composition mimics the non-hematopoietic biological activity of at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO; 3, SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO; 27 AND SEQ ID NO: 30. In another embodiment, the composition is administered by a route selected from the group consisting of orally, buccally, parenterally, nasally, rectally, and topically.

In addition, the present invention provides a method of treating symptoms of a disease, disorder or condition having an inflammatory or autoimmune component in a subject in need thereof, the method comprising the steps: (a) administering to the subject a therapeutically effective amount of a composition comprising at least one stabilized isolated EPO-derived peptide comprising an amino acid sequence comprising an N-terminal end and a C-terminal end and having a non-hematopoietic biological activity; (b)

monitoring the subject's red blood cell indices; and (c) maintaining the subject's red cell indices at substantially normal levels during treatment. In one embodiment of the method, the non-hematopoietic biological activity of the stabilized isolated EPO-derived peptide is stable when the peptide is stored at 4° C. In another embodiment of the method, the isolated stabilized EPO-derived peptide is stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or the C-terminal end of the peptide's amino acid sequence. In another embodiment of the method, the isolated stabilized EPO-derived peptide is at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 10, SEQ ID NO: 14, and SEQ ID NO: 27. In another embodiment of the method, the stabilized isolated EPO-derived peptide is stabilized by a disulfide bond formed between a sulfhydral group of a first amino acid residue and a sulfhydral group of a second amino acid residue along the peptide sequence. In another embodiment of the method, the isolated stabilized EPO-derived peptide comprises at least 6 amino acids having the peptide sequence XAEHYS, wherein X is a first amino acid residue containing a sulfhydral group and Y is a second amino acid residue containing a sulfhydral group, and wherein the sulfhydral group of the first amino acid residue is at an appropriate distance from the sulfhydral group of the second amino acid residue to form the disulfide bond with the sulfhydral group of the second amino acid residue within the peptide sequence, thus stabilizing the peptide. In another embodiment of the method, the isolated disulfide bond-stabilized EPO-derived peptide is at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO; 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, and SEQ ID NO: 15. In another embodiment of the method, the isolated disulfide bond-stabilized EPO-derived peptide is further stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or the C-terminal end of the peptide's amino acid sequence. In another embodiment of the method, the isolated stabilized EPO-derived peptide comprises at least 7 amino acids having the peptide sequence XAEHYS, wherein X is a first amino acid residue containing a sulfhydral group and Y is a second amino acid residue containing a sulfhydral group, and wherein the sulfhydral group of the first amino acid residue is at an appropriate distance from the sulfhydral group of the second amino acid residue to form the disulfide bond with the sulfhydral group of the second amino acid residue within the peptide sequence, thus stabilizing the peptide. In another embodiment of the method, the isolated disulfide bond-stabilized EPO-derived peptide is at least one peptide whose amino acid sequence is SEQ ID NO: 30. In another embodiment of the method, the isolated stabilized EPO-derived peptide is further stabilized by chemically adding a small bicyclic molecule to at least one of the N-terminal end or the C-terminal end of the peptide's amino acid sequence. In another embodiment of the method, the isolated disulfide bond-stabilized EPO-derived peptide is at least one peptide whose amino acid sequence is SEQ ID NO: 20. In another embodiment of the method, the isolated stabilized EPO-derived peptide mimics the non-hematopoetic biological activity of at least one peptide whose amino acid sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO; 3, SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20 AND SEQ ID NO: 30. In yet another embodiment of the method, when administered in a therapeutically effective amount to a subject in need thereof, the stabilized isolated EPO-derived peptide reduces clinical symptoms of at least one disease, disorder, or condition having an inflammatory or autoimmune component selected from the group consisting of an acute cerebrovascular injury, an acute spinal cord injury, an acute brain injury, an acute cardiovascular injury, an arthritis, an autoimmune disease, a demyelinating disease, and immune-mediated inflammation. In still another embodiment, the composition is administered by a route selected from the group consisting of orally, buccally, parenterally, nasally, rectally, and topically.

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